Title:Number of tumor foci predicts prognosis in papillary thyroid cancer

Author's response to reviews Title:Number of tumor foci predicts prognosis in papillary thyroid cancer Authors: Qing-hai Ji (quningfudan@hotmail.com) Ning Qu (jonathan_qn@163.com) Ling Zhang (zhangling@163.com) Yong-xue Zhu (zhuyongxue@163.com) Zhuo-ying Wang (wangzhuoying@163.com) Qiang Shen (shenqiang@163.com) Yu Wang (wangyu@163.com) Duan-shu Li (liduanshu@163.com) Version:2Date:7 October 2014 Author's response to reviews: see over

Dear Senior Editor, Please re-consider our manuscript "Number of tumor foci predicts prognosis in papillary thyroid cancer" for publication in BMC cancer. Thank you very much for meticulously reviewing our previous manuscript. We would also like to thank the reviewers for their thoughtful and constructive comments. In response to the editor s suggestions and the reviewer concerns, we have added more detailed information where appropriate. All of these modifications have vastly improved our manuscript. The significantly revised portions have been marked in blue. We also have our manuscript reviewed by American Journal Experts for English speaking editing. We are pleased to resubmit our revised manuscript to you for further consideration. We hope that the editors and reviewers will be satisfied with our revised manuscript. All authors concur with the submission and the material submitted for publication has not been previously reported and is not under consideration for publication elsewhere. None of the authors have any commercial interest nor do they have conflict of interest. We have disclosed all of our founding sources. Thank you for your consideration. Sincerely yours, Ji Qing-hai M.D. Department of oncology, Shanghai Medical College Department of Head & Neck Surgery, Fudan University Cancer Center

COMMENTS FOR AUTHOR: Reviewer 1: Rondi Kauffmann DISCRETIONARY REVISIONS: 1. Title Page: Would recommend changing title to Number of tumor foci predicts prognosis in papillary thyroid cancer We thank the reviewer for the helpful suggestion, Line 2. 2. Title Page: Capitalize Oncology in Department of Oncology We thank the reviewer for the helpful suggestion, Line 6. 3. Abstract Background: second sentence- evaluated should be evaluate We thank the reviewer for the helpful suggestion, Line 28. 4. Abstract methods: second sentence- remove the period after (3 or more foci) We thank the reviewer for the helpful suggestion. 5. Abstract methods: fourth sentence- relation should be relationship We thank the reviewer for the helpful suggestion, Line 33. 6. Abstract results: third sentence- define the abbreviations for CLNM and LLNM prior to using them in the abstract We thank the reviewer for the helpful suggestion, Line 37-38.

7. Abstract results: fourth sentence- remove for the trend after the p value- it is unnecessary We thank the reviewer for the helpful suggestion. 8. Abstract results: fourth sentence- lowest rate of recurrence should be changed to shortest recurrence-free survival We thank the reviewer for the helpful suggestion, Line 39-40. 9. Abstract results: fifth sentence- this is a difficult sentence to understand, because the structure is a bit unusual and confusing. Would change the sentence to something like Independent predictors of recurrence by multivariate Cox analysis included >3 tumor foci (HR 2.595, CI 1.533-4.393), and extrathryoidal extension (HR 1.947, CI 1.123-3.377). We thank the reviewer for the helpful suggestion, Line 42-44. 10. Abstract conclusion: first sentence- would change beginning of sentence to be something like The presence of multifocal papillary thyroid carcinoma is associated with a tendency.. We thank the reviewer for the helpful suggestion, Line 45-46. 11. Materials and methods- second sentence- there should be a were after the (TT), and when comprised should be comprising. Delete the 496/2115, as it is unnecessary. We thank the reviewer for the helpful suggestion, Line 85. 12. Initial treatment- spell out the meaning of LLND before it is used for the first time We thank the reviewer for the helpful suggestion, Line 100.

13. Initial treatment- first sentence should read an ultrasonographic (US) examination We thank the reviewer for the helpful suggestion, Line 88. 14. Recommend changing sex to gender throughout manuscript. We thank the reviewer for the helpful suggestion, we have changed sex to gender throughout manuscript. 15. Follow-up first sentence- remove of before patients We thank the reviewer for the helpful suggestion. 16. Results- please spell out female:male ratio We thank the reviewer for the helpful suggestion, Line 141. 17. Discussion second paragraph first sentence- remova the could, and predict should be predicted We thank the reviewer for the helpful suggestion, Line 206. 18. Discussion- second paragraph- evolvement should be evolution We thank the reviewer for the helpful suggestion, Line 214. 19. Discussion- third paragraph- was positively should be is positively. Should be poorer prognosis. reported should be reporting. multifocality was associated should be multifocality is associated.

We thank the reviewer for the helpful suggestion, Line 221 and 223. 20. Table 1- change sex to gender We thank the reviewer for the helpful suggestion, Table 1. Minor Essential Revisions: 1. Abstract/manuscript: Because the TNM staging system for papillary thyroid cancer uses N1, N2 designations as a way to describe extent of nodal involvement for purposes of staging, it is confusing in this manuscript that the 3 different groups evaluated are labeled N1 for one tumor focus, N2 for two foci of disease, and N3 for > 3 tumor foci. I would recommend changing the labeling of the groups, to facilitate understanding and address the confusion of using the same label for groups as is used universally for staging this disease. We thank the reviewer for the helpful suggestion, we have changed the labeling of the groups to "G" throughout manuscript. 2. Background first sentence- This sentence is quite confusing, because the structure is unusual. Please reword to facilitate understanding. I would suggest something like With the number of cases increasing annually, it is estimated that there will be 62,980 newly-diagnosed thyroid carcinoma cases in the United States in 2014, more than 90% of which will be papillary thyroid cancer (PTC). We thank the reviewer for the helpful suggestion, Line 49-51. 3. Background final sentence- This sentence is quite long. Would recommend starting new sentence after the references 7 and 8. Also, delineate should be delineating and investigate should be investigating. We thank the reviewer for the helpful suggestion, Line 57-59.

4. Follow-up second sentence- as visiting by callings does not make sense. Does this mean follow-up phone calls to patients? We thank the reviewer for the helpful suggestion, we have revised it to "a follow-up phone call to the patient" (Line 115). 5. Please spell out RFS the first time that the abbreviation is used. We thank the reviewer for the helpful suggestion, Line 129. 6. Results- number of tumor foci and recurrence- The last sentence of the first paragraph is very confusing. Are the authors trying to state that patients with >3 tumor foci have the shortest RFS, followed by N2 and N1? This can be stated more simply. We thank the reviewer for the helpful suggestion, Line 171-172. 7. Results- number of tumor foci and recurrence- last paragraph- The second half of the paragraph is very confusing and wordy. These last 4 sentences should be combined and simplified to reflect that there was an increased risk of recurrence with increasing numbers of tumor foci. Additionally, extrathyroidal tumor extension was an independent predictor of recurrence (HR 1.947). We thank the reviewer for the helpful suggestion, Line 175-181. 8. Table 1 Does the 42.1% of patients with multifocal disease include the 60.1% of patients with bilateral disease? I am confused, because these percentages add up to more than 100%. We thank the reviewer for the helpful suggestion, We thank the reviewer for the helpful suggestion. There is an error in calculating the number of patients with bilateral malignancy. The fact is that 209 (42.1%) patients had multifocal disease, including 178 cases with bilateral

tumors. The other 120 patients with bilateral nodules revealed preoperatively, and were confirmed as unilateral malignancy on pathology. 9. Figure 2: Title should read Kaplan-Meier curves for Cancer Specific Survival (CSS) We thank the reviewer for the helpful suggestion, Figure 2. MAJOR COMPULSORY REVISIONS: 1. (1) In the Initial treatment section, the authors state that FNA was not routinely performed before surgery. As this is an important diagnostic step, and is considered standard of care when evaluating a newly-diagnosed thyroid nodule, the authors should comment on why the standard of care is different in China, compared to what is typically done in the United States. We thank the reviewer for the helpful suggestion. FNAB or US-FNAB is a safe, simple, accurate, and cost-effective diagnostic tool preoperatively. Because our hospital is one of top cancer center in China, a large number of patients all around the country come to visit, generating the problem of insufficient number of radiologists who perform US-FNAB for each patients and cytopathologists who analyze all slides containing FNAB results. Therefore, FNAB or US-FNAB was not routinely performed before surgery. The dilemma have been improved greatly sine 2010, but the present study retrospectively reviewed the cases treated at our center from 1983 to 2007, in which period the proportion of FNAB or US-FNAB preoperatively was still low[1, 2]. (2) Furthermore, a lobectomy with ipsilateral central lymph node dissection was done as initial treatment for papillary thyroid cancer. By what is explained in the manuscript, mention is made of histology of the frozen section (FS) guiding extent of surgical procedures in these patients. Is this to mean that if an intra-operative FS revealed papillary thyroid cancer, a completion thyroidectomy was done at the time of the initial operation, or was this done later? If a completion thyroidectomy was not done when papillary thyroid carcinoma was diagnosed, I wonder about whether additional patients may have actually had multifocal disease (i.e. in the other lobe) that was never diagnosed because no obvious nodules were present.

We thank the reviewer for the helpful suggestion. When undetermined nodules are detected in the contralateral lobe by US at our center, two types of surgeries were performed: a subtotal lobectomy or TT ( if patients older than 45 yr, the primary tumor was greater than 1 cm, and undetermined nodules were detected in the contralateral lobe by US and regional metastases or multifocal tumors were present). When malignant lesions were identified in both lobes of the thyroid by FS after a subtotal lobectomy, a completion thyroidectomy (CT) was done at the time of the initial operation. However, when papillary thyroid carcinoma was found only in unilateral lobe of thyroid by US, intraoperative inspection and intraoperative FS, a completion thyroidectomy would not be done. For these patients, postsurgical physical examinations were performed every 3 6 months. During the follow-up visits, all patients underwent US examinations of the neck. Our follow-up data showed very few patients presented residual tumor in the other lobe[2]. (Line 91-100) (3) Also, because intra-operative FS is notoriously difficult to interpret, what was the false negative rate for malignancy on intra-operative FS? Did patients who had a negative FS in the OR, only to have malignancy shown on final pathology, go back to the OR for completion thyroidectomy? This information is not provided in the manuscript, but is important information both in terms of evaluating adequacy of treatment for thyroid cancer, as well as potential impact on rates of recurrence and disease-specific survival. We thank the reviewer for the helpful suggestion. Intra-operative FS is difficult to interpret for follicular cancer, at our center, the false negative rate for malignancy on intra-operative FS is. It seldom happens that patients who have a negative FS in the OR, in this case, the intra-operative management depends on the preoperative evaluations and intraoperative inspection. If the results of US and other image examinations suggest the lesions with high risk of malignancy, we treat them as malignancy. On the other hand, if the lesions are too small to be found on US or other preoperative examinations, but shown on final pathology (incidentaloma), we would not go back to the OR for completion thyroidectomy, the reasons are as follows: firstly, the incidence of thyroid incidentaloma after thyroidectomy is quite low at our center (<5%); secondly, long-term follow-up data do not show higher rates of recurrence and shorter disease-specific survival than

the common[2]. 2. Follow-up- it is not specified in this section whether biopsy was required to diagnose recurrent or distant disease. A number of imaging studies are mentioned, but please clarify whether tissue biopsy was obtained for confirmation. From the way that the section is written, it sounds as though imaging OR FNA was performed. We thank the reviewer for the helpful suggestion. In present study, the neck recurrence was classified as a new biopsy-proven/secondary surgery-confirmed local disease within the residual thyroid gland or lateral lymph nodes;the distant recurrence was classified as a distant disease revealed by ultrasonography and/or imaging scans located in other sites, including the lungs, bones or brain. Tissue biopsy was obtained for confirmation of neck recurrence. (Line 116-124) 3. Results- The section on baseline characteristics states that the age of patients ranged from 7-85 years. With some patients being this young, were patients with known genetic mutations, or known familial genetic predisposition for thyroid cancer included in this analysis? These individuals may have worse prognosis, by virtue of a genetic mutation, and should not be included with standard papillary thyroid cancer. We thank the reviewer for the helpful suggestion. In present study, there were no patients with known familial genetic predisposition for thyroid cancer. Genetic mutations were not inspected at our center, but this would be a beneficial examination for young patients and might be a good predictor for prognosis except for the traditional clinicopathologic features. 4. Results- In this section, it is stated that 209 (42.1%) patients had multifocal disease, and 298 patients had bilateral (60.1%). Are these groups not one and the same? The percentages add up to more than 100%, so I am unclear which groups these patients fall into. We thank the reviewer for the helpful suggestion. There is an error in calculating the number of patients with bilateral malignancy. The fact is that 209 (42.1%) patients had

multifocal disease, including 178 cases with bilateral tumors. The other 120 patients with bilateral nodules revealed preoperatively, and were confirmed as unilateral malignancy on pathology. 5. Number of foci and survival- This paragraph gives what sounds to be contradictory results: It states that there was a significant trend of decreasing CSS according to number of tumor foci (p value 0.041), but then in the next sentence, states that the differences in CSS were not significant among the three groups (p value 0.087). Where are these p values from? Is one univariate and the other multivariate? What is the difference between the two, since one is statistically significant and the other is not? We thank the reviewer for the helpful suggestion. This is an interpretation by software, the trend of decreasing CSS according to number of tumor foci was tested by log-rank method, and P value was 0.041. However, Kaplan-Meier curves for Cancer-Specific Survival (CSS) of G1, G2 and G3 group had crossed (Figure 2), then it was known that the differences in CSS were not significant among the three groups. After a consultation from a statistician ( Hai-dong Kan, Professor, School of Public Health, Fudan University, Shanghai 200032, China), it was re-understood that in this case, the trend should be tested by log-rank (Mantel-Cox) test, thus the results suggested that both the trend and the differences were insignificant. (Line 188-190) 6. Table 2 shows increased risk of neck recurrence by increasing number of tumor foci, with a statistically significant p value. Yet, the risk of distant recurrence is not predicted by number of tumor foci. The authors do not address in their discussion. I would be interested in the authors interpretation of this data. Are patients dying of locally recurrent papillary thyroid cancer? If they are not recurring distantly or systemically, what is the cause of their disease-specific death? We thank the reviewer for the helpful suggestion. In present series of patients, the risk of neck recurrence increased significantly by increasing number of tumor foci, but the he risk of distant recurrence was not predicted by number of tumor foci. Similar with the risk of distant

recurrence, the risk of disease-specific death was not predicted by number of tumor foci, either. The causes of disease-specific death were locally recurrent cancers (n=2), lung metastases (n=6), bone metastases (n=2), brain metastasis (n=1) and malignant consumptions (n=4). 7. In Table 3, predictors of recurrence are displayed. Age was not a predictor, but it was included as a continuous variable. I would be interested whether age would be predictive if it was included as a categorical variable (<45 years vs. >45), as is a known prognostic factor. We thank the reviewer for the helpful suggestion. When patient age as an binary variable (<45 years vs. >45) was included in multivariate Cox regression for recurrence, it was not associated with recurrence, either (HR 0.802, 95% CI 0.461-1.397, P=0.437). 8. This is a retrospective study. As such, the discussion should include an acknowledgement of the limitations of such a study. There is very little explanation offered by the authors as to the potential limitations of this study or the dataset used. In particular, are co-morbidities accounted for? What are other potential limitations that may inform the data or the results? We thank the reviewer for the helpful suggestion. Though our study was not a randomized case-control trial, we reviewed the medical records dating back to 1983, and we followed a unified in-house protocol to treat PTC in order to recruit as unselected a cohort as possible. Though it is unlike a standard treatment for the PTC patients in the United States, the TT procedure we do allows for the evaluation of tumor multifocality. However, there are several limitations to this study. First of all, we did not find any relationship between the number of tumor foci and distant recurrence or cancer-specific death, the prognostic significance of the number of tumor foci remains in the influence of neck recurrence. We plan to follow this cohort for a longer period of time to evaluate the long-term influence of multifocality on thyroid cancer mortality. Secondly, multifocal-unilateral and multifocal-bilateral PTC were not analyzed as specific subgroups. Further study is recommended to compare the clinicopathologic features and clinical outcomes of these two groups to evaluate the association between tumor location and disease outcomes. At last, genetic mutations were

not examined and investigated for the association with the number of tumor foci and the prognosis in PTC. 9. Needs some language corrections before being published. We thank the reviewer for the helpful suggestion. We have conducted a thorough and careful revision of grammar and spelling, we also have our manuscript reviewed by American Journal Experts for English speaking editing. Reviewer 2: John Yim Major Compulsory Revisions: The actual size of the tumors is not given in the data, only the maximum size. It is well accepted that multifocal microscopic, specially less than 1 cm, PTC is not considered to affect prognosis. This submission should include the actual number (percentage) of tumors that were less than 1 cm, in addition to what the mean and median, and the range of tumor sizes were. The actual sizes would be best graphically represented. For the Kaplan Meier curves there is no explanation of what relationships are being tested to come up with the p-value. We thank the reviewer for the helpful suggestion. In present study, the maximal tumor size was defined the largest diameter for multifocal lesions, including bilateral lesions, that was the maximal macroscopic or microscopic tumor size of the largest lesion was used, as outlined in the American Joint Committee on Cancer seventh edition. The maximal tumor size was the one of the common clinicopathologic factors investigated in previous studies, relating to predicting prognosis in PTC. The mean and median size was 2.31 and 1.58 cm, and the range of size was 0.1-10.0 cm. The actual percentage of tumors that were less than 1 cm was 17.9% (89/496) in this study, only 15 of them had tumors less than 0.5 cm. Recently for these patients, it was reported that the same treatment was recommended as that used for large PTC (>1 cm) if total tumor diameter (TTD) was more than 1 cm[3]. If the TTD falls within within 1 cm and the patient was consistent with other low-risk features. To some extent, TTD should

be regarded as the true tumor size of multifocal microcarcinoma rather than its greatest dimension. However, we did not observed the similar results, probably due to the small number of patients with tumors less than 1 cm. The low percentage of tumors that were less than 1 cm reflected that in present series, maximal tumor size was reasonable to be investigated as a variable for predictor of prognosis, which was consistent with previous studies[3-5]. For the Kaplan Meier curves, figure 1 indicated that that patients with 3 or more tumor foci had the shortest RFS, followed by N1 and N2, and differences were significant among the 3 groups (P=0.001, Log Rank test). Figure 2 indicated that Although a trend of decreasing CSS was found according to the increasing number of tumor foci, the differences of CSS were not significant statistically among the 3 groups (P=0.087, Log Rank test). Minor Essential Revisions: There are too many mistakes in the English language. In the first eight lines of the Abstract alone there are multiple errors: line 27 evaluated should be evaluate, line 30 should have a comma instead of a period, line 31 should be grouped instead of group, line 32 relation should be relationship. CLNM and LLNM are not defined in line 37. The errors continue on page after page. Please have somebody fluent in the English language edit this submission. Why does it say "elderly patients" in line 140? We thank the reviewer for the helpful suggestion. We have conducted a thorough and careful revision of grammar and spelling, we also have our manuscript reviewed by American Journal Experts for English speaking editing. We hope that the editors as well as reviewers can satisfy our answers. Thank you for your consideration. Sincerely yours,

Ji Qing-hai M.D. Department of oncology, Shanghai Medical College Department of Head & Neck Surgery, Fudan University Cancer Hospital References 1. Zhang L, Li H, Ji QH, Zhu YX, Wang ZY, Wang Y, Huang CP, Shen Q, Li DS, Wu Y: The clinical features of papillary thyroid cancer in Hashimoto's thyroiditis patients from an area with a high prevalence of Hashimoto's disease. BMC Cancer 2012, 12:610. 2. Zhang L, Wei WJ, Ji QH, Zhu YX, Wang ZY, Wang Y, Huang CP, Shen Q, Li DS, Wu Y: Risk factors for neck nodal metastasis in papillary thyroid microcarcinoma: a study of 1066 patients. J Clin Endocrinol Metab 2012, 97(4):1250-1257. 3. Zhao Q, Ming J, Liu C, Shi L, Xu X, Nie X, Huang T: Multifocality and total tumor diameter predict central neck lymph node metastases in papillary thyroid microcarcinoma. Ann Surg Oncol 2013, 20(3):746-752. 4. Ito Y, Fukushima M, Kihara M, Takamura Y, Kobayashi K, Miya A, Miyauchi A: Investigation of the prognosis of patients with papillary thyroid carcinoma by tumor size. Endocr J 2012, 59(6):457-464. 5. Machens A, Holzhausen HJ, Dralle H: The prognostic value of primary tumor size in papillary and follicular thyroid carcinoma. Cancer 2005, 103(11):2269-2273.